CN102016028B - The enzymatic compositions of cellulose-less enzyme and for producing its host cell - Google Patents

Abstract

The present invention is provided to produce the recombinant bacterial cell of detergent additives protein.In some embodiments, described cell is bacillus cell.In other embodiments, described cell comprises the genome of the bglC gene containing inactivation and for producing the recombinant nucleic acid of at least one secreting type detergent additives protein.In some preferred embodiments, described secreting type detergent additives protein is protease.The present invention also provides for using described bacterial cell to produce at least one detergent additives method of protein, and the compositions of the cellulose-less enzyme containing at least one detergent additives protein.

Description

The enzymatic compositions of cellulose-less enzyme and for producing its host cell

Technical field

The present invention is provided to produce the recombinant bacterial cell of detergent additives protein.In some embodiments, This cell is that bacillus cereus (Bacillus) belongs to cell.In other embodiments, described cell comprises containing inactivation The genome of bglC gene and for producing the recombinant nucleic acid of at least one secreting type detergent additives protein.One In a little preferred embodiments, described secreting type detergent additives protein is protease.It is described that the present invention also provides for use Bacterial cell produces at least one detergent additives method of protein and containing at least one detergent additives egg The cellulose-less enzymatic compositions of white matter.

Background technology

Expression and the restructuring generation of allogenic polypeptide are widely used technology.Widely known, can be by coding purpose external source The nuclear transformation cell of polypeptide, is used for expressing and produce substantial amounts of required polypeptide.In some applications, the method is used for producing ratio The more polypeptide of amount naturally-produced in source organism.It is true that the expression of exogenous nucleic acid sequences and the table excessively of endogenous sequence Reach and be widely used in modern biotechnology.

In some cases, it is undesirable to product produce with destination protein matter.Such as, recombinase is being produced (such as Protease, amylase etc.) time, may also produce other less desirable enzymes (such as cellulase).

Although molecular biology and protein engineering are achieved with progress, but remain a need for may be used for reducing (if not disappearing If removing) these undesirably active method and compositions.

Summary of the invention

The present invention is provided to produce the recombinant bacterial cell of detergent additives protein.In some embodiments, This cell is bacillus cell.In other embodiments, described cell comprises the base of the bglC gene containing inactivation Because of group, and for producing the recombinant nucleic acid of at least one secreting type detergent additives protein.Preferably implement at some In scheme, described secreting type detergent additives protein is protease.The present invention also provides for using described bacterial cell to produce At least one detergent additives method of protein raw and containing at least one detergent additives protein without fiber Element enzymatic compositions.

The present invention provides the Bacillus spec host cell of restructuring, and it comprises the gene of the bglC gene containing inactivation Group, wherein this cell also comprises the recombinant nucleic acid for producing secreting type detergent additives protein.The most real at some Executing in scheme, the bglC gene of described inactivation contains disappearance, inserts, replaces or reset.In other preferred embodiments, Described Bacillus spec cell is Bacillus licheniformis (B.licheniformis), bacillus subtilis (B.subtilis), Bacillus clausii (B.clausii), Alkaliphilic bacillus (B.alkalophilus) or salt tolerant spore The cell of bacillus (B.halodurans).In other preferred embodiments, described secreting type detergent additives albumen Matter is the enzyme selected from protease, amylase, pectate lyase and lipase.In some particularly preferred embodiments, should Enzyme is protease.In some more particularly preferred embodiments, described protease is subtilisin.Excellent at other In the embodiment of choosing, described bglC gene code and SEQ ID NO:2 have the polypeptide of at least 80% homogeneity.

The present invention also provides for comprising culture medium and the cell culture of recombinated bacillus species host cell, described place Chief cell comprises the genome of bglC gene containing inactivation, and wherein said cell also comprises and adds for producing secreting type detergent Add the recombinant nucleic acid of agent protein.In some preferred embodiments, the bglC gene of described inactivation contain disappearance, insertion, Replace or reset.In other preferred embodiments, described Bacillus spec cell is Bacillus licheniformis (B.licheniformis), bacillus subtilis (B.subtilis), Bacillus clausii (B.clausii), basophilic spore Bacillus (B.alkalophilus) or the cell of salt tolerant bacillus cereus (B.halodurans).In other preferred embodiments In, described secreting type detergent additives protein is selected from protease, amylase, pectate lyase, acyltransferase, virtue The enzyme of base esterase and lipase.In some particularly preferred embodiments, this enzyme is protease.The most preferred at some In embodiment, described protease is subtilisin.In other preferred embodiments, described bglC gene is compiled Code and SEQ ID NO:2 have the polypeptide of at least 80% homogeneity.

The present invention also provides for being included under conditions of being suitable to produce described secreting type detergent additives protein and remains thin The method of born of the same parents' culture.In some embodiments, the method also includes that reclaiming described secreting type detergent from culture medium adds Add agent protein.In other embodiments, the method also include by described secreting type detergent additives protein with wash Clothing detergent (laundry detergent) combines.In other preferred embodiments, described detergent additives albumen Matter is subtilisin.

The present invention also provides for the protein of cellulose-less enzyme or the enzymatic compositions produced by methods described herein.At some In preferred embodiment, described compositions comprises the secreting type detergent additives albumen produced by Bacillus spec Matter, and it is characterized in that said composition does not have detectable cellulase activity.

The present invention also provides for comprising the nothing fibre of the secreting type detergent additives protein produced by methods described herein Dimension element enzyme laundry detergent.In some embodiments, the laundry detergent of described cellulose-less enzyme comprises cellulosic polymerization Thing (cellulosic polymer).

The present invention also provides for the method for producing host cell, and it includes the first recombinant nucleic acid is introduced bacillus In Species Cell, so that this recombinant nucleic acid and the bglC gene recombinaton of this cell；And in this cell, introduce the second recombinant nuclear Acid, to provide secreting type detergent additives protein expression.In some preferred embodiments, described nucleic acid inserts In bglC gene.In other preferred embodiments, described nucleic acid causes the disappearance of at least one part of bglC gene.? In other preferred embodiments, described secreting type detergent additives protein is subtilisin.

Accompanying drawing is sketched

Some aspect following detailed description of can be fully understood after reading in conjunction with the accompanying.It is emphasized that root According to general practice, multiple feature not drawn on scale of accompanying drawing.On the contrary, the size of multiple features for clarity sake and is subjectively entered Go and zoomed in or out.Accompanying drawing includes following figure:

Figure 1A-B provides the strategy of the Bacillus host cell for building the bglC gene containing inactivation.

Fig. 2 shows the bacillus subtilis strain (i.e. " host A " and " host B ") at two kinds of blgC genes with inactivation And the culture supernatants of the bacillus subtilis strain (i.e. " host C " and " host D ") of two kinds of bglS genes with inactivation On carry out viscosimetric analysis test result.Fluid viscosity is weighed with centipoise (i.e. cP).

Fig. 3 shows in the culture supernatants of two kinds of bacillus subtilis strains producing recombination bacillus subtilis protease The result of the viscosimetric analysis test carried out.Bacterial strain MDT-05-28 has the bglC gene of inactivation, and bacterial strain MTD-04-250 contains Wild type bglC gene.

Detailed Description Of The Invention

The present invention is provided to produce the recombinant bacterial cell of detergent additives protein.In some embodiments, Described cell is bacillus cell.In other embodiments, described cell contains the bglC gene that comprises inactivation Genome and for producing the recombinant nucleic acid of at least one secreting type detergent additives protein.Preferably implement at some In scheme, described secreting type detergent additives protein is protease.The present invention also provides for using this bacterial cell to produce At least one detergent additives method of protein and the cellulose-less containing at least one detergent additives protein Enzymatic compositions.

Except as otherwise noted, otherwise the enforcement of the present invention relates to molecular biology, microbiology, protein purification, albumen Normally used routine techniques in matter engineering, protein and DNA sequencing and recombinant DNA field, these technology belong to this area Technology category.These technology are known to a person skilled in the art, and are described in a large amount of textbook and list of references (refering to such as Sambrook etc., " Molecular Cloning:A Laboratory Manual ", the second edition (Cold Spring Harbor), [1989])；And Ausubel etc., " Current Protocols in MolecularBiology " [1987]). All patents, patent application, article and the publication mentioned above and below herein is incorporated to the most in way of reference Herein.

Additionally, title provided herein is not constituted to various aspects of the present invention or the restriction of embodiment, described side Face and embodiment can be explained with reference to book entirety and obtain.Therefore, the term defined immediately below will be based on whole explanation Book more fully defines.But, for the ease of understanding the present invention, multiple term is defined below.

Definition

Unless otherwise indicated herein, all scientific and technical terminologies the most used herein are respectively provided with of the art general The implication that logical technical staff is generally understood that.Such as, Singleton and Sainsbury, Dictionary of Microbiology And Molecular Biology, the second edition, John Wileyand Sons, NY (1994) and Hale and Marham, The Harper Collins Dictionaryof Biology, Harper Perennial, NY (1991) are those skilled in the art Provide the general dictionary of many terms used herein.Although any method similar or equivalent with methods described herein and material It is used equally to implement the present invention with material, but this document describes preferred method and material.Therefore, term defined below will ginseng Examine description entirety and be able to more complete description.Meanwhile, noun used herein all includes plural reference, unless it is the most civilized Really expression is not so.It is therefoie, for example, include multiple such host cell when mentioning " host cell ".Equally, " base is mentioned Cause " time include multiple such candidate agent, include one or more cells and those skilled in the art when mentioning " cell " Mentioning of its equivalent known, the rest may be inferred.

Numerical range comprises the numerical value defining this scope.It is true that each the highest numerical value be given in this specification Boundary is intended to include each relatively low numerical limits, as the most clearly write out these relatively low numerical limits.This The each minimum numerical limits be given in description full text is by including each higher numerical limits, as the most clearly write out These high value boundaries are such.The each numerical range be given in this specification all includes falling into these wider numerical value In the range of each narrower numerical range, as the most clearly write out in these narrower numerical rangies.When giving When going out numerical range, it should be appreciated that also specifically disclose interval lower limit unit between the upper limit of this scope and lower limit very One of each intermediate value, unless it is not so that its context clearly indicates.These small range of upper limits and lower limit can be independent Ground includes or not included in this scope, and these include one, two end points or do not include that the smaller range of end points is also wrapped Containing in the present invention, and get rid of the boundary of any concrete eliminating in described scope.When described scope includes one or two end points Time, the scope getting rid of one or two end points being included is also included in the present invention.

In the relevant portion that all documents quoted all are expressly incorporated herein by reference, any document is quoted and the most should not manage Solve as to recognize that it is the prior art of the present invention.Any content herein is not necessarily to be construed that and recognizes that the present invention is not eligible for making For formerly invention prior to these publications.All patents mentioned above and publication (include in these patents and publication Disclosed all sequences) the most clearly it is incorporated herein.

Although being used equally to implement or test this with similar or equivalent any suitable method described herein and material Bright, but exemplary and preferred method and material now will be described.

Except as otherwise noted, otherwise nucleic acid is write from left to right with the direction of 5 ' to 3 ', and aminoacid sequence is then with amino Direction to carboxyl is write from left to right.Title provided herein is not constituted to various aspects of the present invention or embodiment Limiting, described aspect and embodiment can be explained with reference to book entirety and obtain.It should be understood that the present invention is not only restricted to be retouched Concrete grammar, scheme and the reagent stated, they can use their situation to change according to those skilled in the art.Therefore, under The term of literary composition definition more completely should define with reference to description entirety.

Terms used herein " is recombinated " and is referred to the most naturally occurring polynucleotide or polypeptide in host cell.Recombinant molecule Can be containing by two or more native sequences linked together in non-natural mode.Reconstitution cell contains the many nucleoside of restructuring Acid or polypeptide.

Terms used herein " allos " refers to the most incoherent natural element.Such as, if host cell produces allos Albumen, then this albumen is not the protein that this host cell is naturally-produced.Similarly, be effectively connected with allogeneic coding sequence opens Mover refers to and the promoter generally effectively not connected with its coded sequence being effectively connected in wild-type host cells.

When for polynucleotide or protein, terms used herein " homology " refers in host cell naturally occurring many Nucleotide or protein.

Term " protein " and " polypeptide " are used interchangeably herein.

" signal sequence " used herein is the aminoacid sequence that protein N terminal part exists, and it is conducive to this albumen The mature form of matter is secreted into outside cell.The definition of signal sequence is functional one.The exoprotein of mature form lacks Few signal sequence, this signal sequence is cut in secretion process.

" coded sequence " used herein is the region of DNA section of coded polypeptide.

" gene of inactivation " used herein is the locus in genome, and it can produce protein (i.e. before inactivation Can be transcribed into and can translate the RNA producing full-length polypeptide).For the gene of codase, when it can not be transcribed and translate into During the catalysis reactive protein of total length, it it is inactivation.Such as can change by changing the sequence needed for genetic transcription and inactivated gene Become the sequence needed for RNA processing (as polyA tail adds), or change the sequence needed for translation.The example of the gene of inactivation includes But be not limited only to the gene of disappearance, gene containing absent region, containing resetting the gene in region, containing inactivating-point mutation or shifting The gene of code, and containing the gene inserted.Further, it is also possible to use antisense or any other can eliminate the side of gene expression Method carrys out inactivated gene.

Terms used herein " nucleic acid " includes DNA, RNA of strand or double-strand, and include through chemical modification DNA or RNA.Term " nucleic acid " and " polynucleotide " are used interchangeably herein.

Terms used herein " carrier " refers to the polynucleotide being designed for that nucleic acid introduces one or more host cells. In some preferred embodiments, carrier independently replicates in different hosts cell.This term is intended to include but are not limited to Cloning vehicle, expression vector, shuttle vector, plasmid, phage particle, box etc..

" expression vector " used herein refers to comprise the DNA of the protein coding region being effectively connected with suitable control sequence Construct, described control sequence can realize the expression of this albumen in suitable host cell.In some embodiments, so Control sequence include realizing transcribing promoter, control to transcribe and produce the optional operator sequence of mRNA, coding mRNA On the sequence of suitable ribosome binding site, enhancer and control transcription and translation terminate sequence.

Terms used herein " promoter " refers to the regulation sequence that initial downstream nucleic acid is transcribed.

Terms used herein " effectively connection " refers to arrange element in the way of allowing element to be functionally correlated with.Such as, If promoter controls transcribing of coded sequence, then promoter is effectively connected with this coded sequence.

Terms used herein " selected marker " refers to such protein, and it can be expressed consequently facilitating select to contain in host There are the nucleic acid of introducing or the host of carrier.The example of selected marker includes but are not limited to antimicrobial and (such as hygromycin, wins Mycin or chloromycetin) and/or the gene of imparting host cell metabolism advantage (such as nutritional advantages).

Terms used herein " from " contain term " come from ", " deriving from " or " being available from " and " being isolatable from ".

" avirulence " used herein biology is to people and/or the biology of other animal no pathogenicities.Used herein Term " reclaims ", " separation " and " separately " refers to protein, cell, nucleic acid or aminoacid natural relevant to it from least one Component removes.

When for cell, it is non-that terms used herein " converts ", " stable conversion " and " transgenic " refers to that this cell has Natural (such as allos) nucleotide sequence, described non-native nucleic acid sequence is integrated in its genome or with the shape of additive type plasmid Formula exists, and goes through many generations and maintain.

Terms used herein " expresses " process referring to that nucleotide sequence based on gene produces polypeptide.This process includes transcribing And translation.

In the context that nucleotide sequence is inserted cell, terms used herein " introduce " refer to " transfection ", " conversion " or " transduceing ", and include being integrated into by nucleotide sequence in eucaryon or prokaryotic cell, its amplifying nucleic acid can be integrated into cellular genome (such as Chromosome, plasmid, plastid or mitochondrial DNA) in, change into autonomous replicon or transient expression (such as the mRNA of transfection).

Terms used herein " hybridizes " mistake referring to that nucleic acid chains is combined with complementary strand by base pairing known in the art Journey.If a nucleic acid hybridizes, then under medium paramount stingent hybridization and wash conditions the most specifically with reference to nucleotide sequence Think that this nucleic acid " optionally can hybridize " with reference to nucleotide sequence.Medium and high stringent hybridization condition is those skilled in the art Known (refer to such as Ausubel etc.,ShortProtocols in Molecular Biology.The third edition, Wiley&Sons, Hoboken, NJ [1995]；And Sambrook etc.,Molecular Cloning:A Laboratory Manual,The third edition, Cold Spring Harbor, NY [2001]).One example of high stringent condition be included in about 42 DEG C 50% Methanamide, 5 × SSC, 5 × DenhardtShi liquid, 0.5%SDS and 100 μ g/ml modified support DNA hybridize, thereafter room temperature with 2 × SSC and 0.5%SDS washes twice, and 42 DEG C wash twice with 0.1 × SSC and 0.5%SDS again thereafter.

Cellulose that terms used herein " cellulosic polymer " refers to comprise at least one Isosorbide-5-Nitrae-β-D glycosidic bond, half Cellulose or the cellulose of modification or hemicellulose polymer.

Terms used herein " cellulose " refers to comprise the polysaccharide polymer of the glucose residue bonded by β-Isosorbide-5-Nitrae.

It is residual by non-glucose sugar bonded for β-(1-4) that terms used herein " hemicellulose " refers to comprise at least one The polysaccharide polymerization of base (such as xylose, galactose, arabinose, rhamnose, mannose, alduronic acid or galacturonic acid or xylan) Thing.Hemicellulose include xylan, glucuronoxylan, araboxylan, arabinogalactan, glucomannan, Xyloglucan and galactomannan.

" cellulase " used herein refers to the Isosorbide-5-Nitrae-β-D-glucosides in hydrocellulose, lichens and corn callose The enzyme of key.Cellulase as herein described has the activity being described as EC 3.2.1.4 according to IUMBM enzyme nomenclature.Fibre described herein The entitled Isosorbide-5-Nitrae of system-(1,3:1,4)-callose 4-glucan hydrolase of dimension element enzyme.

Terms used herein " Bacillus spec " (or bacillus cereus) (such as Bacillus host cell) refers to appoint What Bacillus spec, includes but are not limited to bacillus subtilis (B.subtilis), Bacillus licheniformis (B.licheniformis), bacillus lentus (B.lentus), bacillus brevis (B.brevis), stearothermophilus spore bar Bacterium (B.stearothermophilus), Alkaliphilic bacillus (B.alkalophilus), bacillus amyloliquefaciens (B.amyloliquefaciens), Bacillus clausii (B.clausii), salt tolerant bacillus cereus (B.halodurans), huge Bacterium anthracoides (B.megaterium), Bacillus coagulans (B.coagulans), Bacillus circulans (B.circulans), B.lautus and bacillus thuringiensis (B.thuringiensis), and their subspecies.

" bacillus cereus of cellulose-less enzyme " used herein refers to the Bacillus host cell through genetic modification, its regardless of Secrete the cellulase of detectable amount.As being discussed in detail below, in certain embodiments, the bacillus cereus of cellulose-less enzyme Bacterial strain can be containing the bglC gene of inactivation.

" the unchanged Bacillus strain of equivalent " used herein refers to such host strain, and it is except bglC gene Do not change (being wild type) identical with the Bacillus strain of cellulose-less enzyme the most in other respects.

" cultivation " used herein refers to cultivating microorganism cell mass in liquid or solid culture medium under optimum conditions.? In some embodiments, cultivate and refer to that fermentable recombinant production purpose foreign protein or other desired end-products are (typically at container Or reactor produces).

" detergent additives protein " used herein refers to the protein in laundry detergent to be added.Detergent adds Agent protein can be that enzyme is (such as protease, amylase, pectate lyase, lipase, acyltransferase, arylesterase or not There is the protein of enzymatic activity).In some particularly preferred embodiments, beta glucan hydrolytic enzyme (i.e. has according to IUMBM enzyme Nomenclature is described as the enzyme of the activity of EC3.2.1.8, EC 3.2.1.32, EC 3.2.1.72, EC 3.2.1.136) arranged especially In addition at term " detergent additives protein ".In other particularly preferred embodiments, xylanase (i.e. has The enzyme of the activity of EC 3.2.1.75 it is described as according to IUMBM enzyme nomenclature) also it is particularly intended to exclude at term " detergent additives egg White matter " outside.

Description full text it may also occur that other terms define.

Before describing exemplary in detail, it should be appreciated that the present invention is not only restricted to the specific embodiment party described Case because they yes can change.Be also to be understood that terms used herein is only used for describing specific embodiments Purpose, it is no intended to limit.

Host cell

As above-mentioned, the invention provides the bacillus cereus host producing the cellulase reducing (as undetectable) level Cell.It is said that in general, the Bacillus host cell of the present invention typically produces thin less than the wild type Bacillus host of equivalent The fiber of about 50% (the most less than about 40%, less than about 30%, less than about 20% or less than about 10%) of the cellulase of born of the same parents Element enzyme.In some embodiments, the cellulase that cell of the present invention produces is less than the fibre of the Bacillus host cell of equivalent About the 5% of dimension element enzyme.In some particularly preferred embodiments, cellulase is (that is, this bacillus cereus place that can't detect Chief cell is the Bacillus host cell of cellulose-less enzyme).Cellulase activity is intended to by any suitable known method Measure, such as by contain with congo red staining cellulose LP agar plate (refering to such as Wolf etc., Microbiol., 141:281-290 [1995]；And Carder, Anal.Biochem., 153:75-9 [1986]) and/or use viscosimetric analysis examination Test (such as following more detailed description) to measure.

In some embodiments, produce cellulase production amount by the bglC gene product expression of minimizing cell to subtract Few Bacillus host cell.In such embodiments, it is possible to use any proper method reduces bacillus cereus host BglC in cell expresses, the method that said method includes but are not limited to such as use antisense molecule or ribozyme.Excellent at some In the embodiment of choosing, reduced the expression of bglC by the bglC gene in inactivated cells.

The DNA sequence of several bacillus cereus bglC genes and the protein of these coded by said gene the most after measured, and are protected It is stored in the Genbank data base of NCBI.This sequence presented below:

ATGAAACGGTCAATCTCTATTTTTATTACGTGTTTATTGATTACGTTATTGACAATGGGCGGCATGATAGCTTCGCC GGCATCAGCAGCAGGGACAAAAACGCCAGTAGCCAAGAATGGCCAGCTTAATAAAAGGTACACAGCTCGTTAACCGA GACGGTAAAGCGGTACAGCTGAAGGGGATCAGTTCACACGGATTGCAATGGTATGGAGAATATGTCAATAAAGACAG CTTAAAATGGCTGAGAGATGATGGGTATCACCGTTTTCCGTGCAGCGATGTATACGGCAGATGGCGGTTATATTGAC AACCCGTCCGTGAAAAATAAAGTAAAAGAAGCGGTTGAAGCGGCAAAAGAGCTTGGGATATATGTCATCATGATGGC ATATCTTAAATGACGGTAATCCAAACCAAAATAAAGAGAAGGCAAAAGAATTCTTCAAGGAAATGTCAAGCCTTTAC GGAAACACGCCAAACGTCATTTATGAAATTGCAAACGAACCAACGGGATGTGAACTGGAAGCGTGATATTAAACCAT ATGCGGAAGAAGTGATTTCAGTTATCCGCAAAAATGATCCAGACAACATCATCATTGTCGGAACCGGTACATGGAGC CAGGATGTGAATGATCTGCGATGACCAGCTAAAAGATGCAAACGTTATGTACGCACTTCATTTTTATGCCGGCACGC ACGGCCAATTTTTACGGGATAAAGCAAACTATGCACTCAGCAAAGGAGCACCTATTTTTTGTGACGAGTGGGAACAA GCGACGCGTCTGGCAATGGCGGTGTATTCCTTGATCAATCGAGGGAATGGCTGAAATATCTCGACAGCAAGACCATT AGCTGGGTGAACTGGAATCTTTCTGATAAGCAGGAATATCCTCGCTTTAAAGCCGGGGGCATCTAAAACAGGCGGCT GGCGGTTGTCAGATTTATCTGCTTCAGGAACATTCGTTAGAGAAAACATTCTCGGCACCAAAGATTCGACGAAGGAC ATTCCTGAACGCCATCAAAGATAAACCCACACAGGAAAATGGTATTTCTGTACAGTACAGAGCAGGGGATGGGAGTA TGAACAGCAACCAAATCCGTCCGCAGCTTCAAATAAAAAATAACGGCAATACCACGGTGATTTAAAGATGTCACTGC CCGTTACTGGTATAAAGCGAAAAACAAAGGCCAAAACTTTGACTGTGACTACGCGCAGATTGGATGCGGCAATGTGA CACACAAGTTTGTGACGTTGCATAAACCAAGCAAGGTGCGATACCTATCTGGAACTTGGATTTAAAAACGGAACGTT GGCACCGGGAGCAAGCACAGGGAATATTCAGCTCCGTCTTCACAATGATGACTGGAGCAATTATGCACAAAGCGGCG ATATTCCTTTTTAAATCAAATACGTTTAAAACAACGAAAAAAATCACATTATATGATCAAGGAAAACTGATTTGGGG AACAGAACCAAATTAG (SEQ ID NO:1)

It it is below the aminoacid sequence of SEQ ID NO:1 coding.

MKRSISIFITCLLITLLTMGGMIASPASAAGTKTPVAKNGQLSIKGTQLVNRDGKAVQLKGISSHGLQWYGEYVNKD SLKWLRDDWGITVFRAAMYTADGGYIDNPSVKNKVKEAVEAAKELGIYVIWHILNDGNPNQNKEKAKEFFKEMSSLY GNTPNVIYEIANEPNGDVNWKRDIKPYAEEVISVIRKNDPDNIIIVGTGTWSQDVNDAADDQLKDANVMYALHFYAG THGQFLRDKANYALSKGAPIFVEGTSDASGNGGVFLDQSREWLKYLDSKTISWVNWNLSDKQESSSALKPGASKTGG WRLSDLSASGTFVRENILGTKDSTKDIPETPSKDKPTQENGISVQYRAGDGSMNSNQIRPQLQIKNNGNTTDLDVTA RYWYKAKNKGQNFDCDYAQIGCGNVTHKFVTLHKPKQGADTYLELGFKNGTLAPGASTGNIQLRLHNDDWSNYAQSG DYSFFKSNTFKTTKKITLYDQGKLIWGTEPN (SEQ ID NO:2)

Additionally, identified several conserved domains of cellulase and a large amount of conservative amino acid, this makes People can identify other bacillus cereuss bglC gene and albumen by bioinformatics method.

In some embodiments, bacillus cereus bglC gene comprises and is stored in NCBI Genbank number with provided above According to the bglC sequence (SEQ ID NO:1) at least 70% (for example, at least 80%, at least 90%, at least 95%, at least 97% in storehouse Or at least 98%) sequence iden.In still further embodiments, bacillus cereus bglC gene under strict conditions with preservation BglC sequence or SEQ ID NO:1 hybridization in NCBIGenbank data base.In other embodiments, bacillus cereus BglC gene code has the sequence of at least 70% with the bglC sequence or SEQ ID NO:2 that are stored in NCBI Genbank data base (sequence of for example, at least 80%, at least 90%, at least 93%, at least 95%, at least 97% or at least 98% is same for row homogeneity Property) polypeptide.The exemplary bglC albumen and the nucleotide sequence that are stored in NCBI Genbank data base include GID:3100136 (Bacillus licheniformis), GID:52348343 (Bacillus licheniformis), GID:42491106 (bacillus amyloliquefaciens) and GID: 50812243 (bacillus subtilises).Above-mentioned Genbank accession number (includes nucleic acid therein and protein sequence and these sequences The annotation of row) all by with reference to being integrally incorporated herein.

In some preferred embodiments, Bacillus host cell is any following species: Bacillus licheniformis, slow Bacillus cereus, bacillus subtilis, bacillus amyloliquefaciens, bacillus brevis, bacstearothermophilus, basophilic spore bar Bacterium, Bacillus coagulans, Bacillus circulans, Bacillus pumilus (B.pumilus), bacillus thuringiensis, gram Lloyd's's bud Spore bacillus or bacillus megaterium.B. subtilis host cell includes but are not limited to: United States Patent (USP) 5,264,366 and 4, Those described in 760,025 (RE 34,606), and 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753 to PB1758, PB3360, JH642,1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DEI00 (ATCC39,094), GX4931, PBT 110 and PEP 211 bacterial strain (refering to such as Hoch etc., Genetics73: 215-228[1973]；United States Patent (USP) No.4,450,235；United States Patent (USP) No.4,302,544 and EP 0134048 (referring also to Palva etc., Gene 19:81-87 [1982]；And Fahnestock and Fischer, J.Bacteriol., 165:796-804 [1986]；With Wang etc., Gene 69:39-47 [1988]).

In some embodiments, host cell includes containing expression cassette (i.e. promoter, coding detergent additives albumen The polynucleotide of matter and transcription terminator) recombinant nucleic acid, wherein said expression cassette be enough to make Bacillus host cell produce Raw detergent additives protein.In some embodiments, described recombinant nucleic acid is integrated in host cell gene group, and In other embodiments, in the autonomous carrier replicated outside described recombinant nucleic acid is present in independent of genome.One In a little embodiments, the polynucleotide of coding detergent additives protein are for express this albumen in Bacillus host cell Matter and carried out codon optimized.

In some particularly preferred embodiments, Bacillus host cell is transformed into and makes protein expression maximum Change.Therefore, in some embodiments, host cell at least one following gene containing inactivation change: degU, degS, DegR and/or degQ is (refering to Msadek etc., J.Bacterid., 172:824-834 [1990]；With Olmos etc., Mol.Gen.Genet., 253:562-567 [1997]).In some particularly preferred embodiments, host cell be with The bacillus subtilis that degU32 (Hy) suddenlys change.In other embodiments, Bacillus host cell is in following gene Containing sudden change and/or disappearance: scoC4 (refering to Caldwell etc., J.Bacteriol .183:7329-7340 [2001])； SpoIIE (refering to Arigoni etc., Mol.Microbiol., 31:1407-1415 [1999])；Its in oppA or opp operon His gene (refering to Perego etc., Mol.Microbiol., 5:173-185 [1991]).It is in fact possible to expect, opp operon In any cause the sudden change of identical phenotype all will to can be used for the Bacillus strain of the change of the present invention with oppA gene mutation In some embodiments.In some embodiments, these sudden changes individually occur, and in other embodiments, exist prominent The combination become.In some embodiments, the bacillus cereus of the change of the present invention is available from one or more said gene In contain the Bacillus host strains of sudden change.In other embodiments, the bacillus cereus of the change of the present invention is permissible It is modified to comprise the sudden change of one or more said gene further.

The Bacillus host cell using any method easily to build is used equally to the present invention, including by cell BglC gene in carry out inserting, lack, replace, frameshit, point mutation and/or reset and change this bglC gene order and build Cell, can be used for the present invention.In some embodiments, Gene Partial to be changed is in coding region, or compiles expressing In regulating element needed for code district.In some embodiments, gene this regulation or control sequence be promoter sequence or its Functional portions (i.e. part necessary to gene expression).Such gene inactivation method is known in the art (refering to such as The Microbiol. such as Wolf, 141:281-290 [1995]).

In some embodiments, host cell constructed as below: recombinant nucleic acid is introduced in bacillus cell, so that should Recombinant nucleic acid and the bglC gene recombinaton in cellular genome；The second recombinant nucleic acid is introduced in cell, to provide secreting type Detergent additives protein expression.In some embodiments, nucleic acid is inserted in bglC gene, and implement at other In scheme, this nucleic acid causes at least some of disappearance of bglC gene.

It is well known to those skilled in the art (ginseng for polynucleotide sequence being introduced the appropriate method of bacillus cell Read such as Ferrari etc., " Genetics, " in Harwood etc. (ed.),Bacillus, Plenum Publishing Corp. [1989], 57-72 page；Referring also to Saunders etc., J.Bacteriol., 157:718-726 [1984]；Hoch etc., J.Bacteriol., 93:1925-1937 [1967]；Mann etc., Curr.Mierobiol., 13:131-135 [1986]；And Holubova, FoliaMicrobiol., 30:97 [1985]；Chang etc., Mol.Gen.Genet, 168:11-115；[1979]； Vorobjeva etc., FEMS Microbiol.Lett., 7:261-263 [1980]；Smith etc., Appl.Env.Microbiol., 51:634 [1986]；Fisher etc., Arch.Microbiol., 139:213-217 [1981]；And McDonald, J.Gen.Microbiol., 130:203 [1984]).It is true that methods such as such as conversions, including protoplast transformation and Congression, transduction and protoplast fusion, be to it known in the art, and be applicable to the present invention.

As noted above, reducing in addition to the cellulase of (such as can't detect) except producing level, the present invention provides Also comprise the Bacillus host cell of recombinant nucleic acid for producing secreting type detergent additives protein, wherein secreting type Detergent additives protein is the protein (such as enzyme) being secreted from cell and being added in laundry detergent.Show The detergent additives protein of example includes but are not limited to protease (such as subtilisin), α-amylase, manna Dextranase, cellulase, lyase, acyltransferase, arylesterase and lipase etc..In some embodiments, detergent Additive protein can be expressed by the bacterial strain identical with the source bacterial strain of this detergent additives protein.

Enzyme

Subtilisin (i.e. the outer alkaline serine protease of born of the same parents) is especially interesting.Any suitable hay bar Mycoproteinase is used equally to the present invention (refering to such as Siezen, Protein Sci., 6:501-523 [1997]；Bryan, Biochim.Biophys.Acta, 1543:203-222 [2000]；Maurer, Curr.Op, Biotechnol., 2,004 15: 330-334[2004]；And Gupta, Appl.Mierobiol.Biotechnol., 59:15-32 [2002]).Implement at some In scheme, purpose subtilisin has the activity being described as EC 3.4.4.16 according to IUMBM enzyme nomenclature.

In some embodiments, such subtilisin has the aminoacid sequence being found in wild type gene group Row (subtilisin that i.e. this subtilisin is naturally-occurring), and in other embodiments, described withered The variant of the subtilisin that grass Bacillus protease is naturally-occurring.In some preferred embodiments, described variant Subtilisin comprise have at least 80% with the subtilisin coded by wild type gene group, at least 90%, The aminoacid sequence of at least 95% or at least 98% homogeneity.Exemplary subtilisin includes but are not limited to:(Novozymes)、FNA^TM(Genencor)、(Novozymes)、 PURAFECT^TM(Genencor)、KAP^TM(Kao)、EVERLASE^TM(Novozymes)、PURAFECT OxP^TM(Genencor)、 FN4^TM(Genencor)、BLAP S^TM(Henkel)、BLAP X^TM(Hen kel)、(Novozymes)、 KANNASE^TM(N ovozymes) and PROPERASE^TM(Genencor).In other embodiments, described bacillus subtilis egg White enzyme includes but are not limited to subtilisin 168, subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin 147 or subtilisin 309 are (refering to such as EP414279B；WO89/06279；With Stahl etc., J.Bacteriol., 159:811-818 [1984]).Other can be used for this Bright subtilisin and other protease include but are not limited to those described in documents below: WO 99/20770； WO 99/20726；WO 99/20769；WO89/06279；RE 34,606；United States Patent (USP) No.4,914,031；United States Patent (USP) No.4,980,288；United States Patent (USP) No.5,208,158；United States Patent (USP) No.5,310,675；United States Patent (USP) No.5,336,611； United States Patent (USP) No.5,399,283；United States Patent (USP) No.5,441,882；United States Patent (USP) No.5,482,849；United States Patent (USP) No.5, 631,217；United States Patent (USP) No.5,665,587；United States Patent (USP) No.5,700,676；United States Patent (USP) No.5,741,694；The U.S. is special Profit No.5,858,757；United States Patent (USP) No.5,880,080；United States Patent (USP) No.6,197,567；And United States Patent (USP) No.6, 218,165。

Detergent and detergent additives protein

In some embodiments, host cell is used to prepare protein compositions and laundry detergent, wherein at some In preferred embodiment, described detergent contains at least one cellulosic polymer.

In some embodiments, host cell is cultivated to provide at least one secreting type detergent additives protein to enter Enter to cultivate in the culture medium of cell.In some particularly preferred embodiments, use any suitable method (as precipitation, from The heart, affine, filtration or any other means known in the art) from culture medium, reclaim described secreting type detergent additives albumen Matter.It is, for example possible to use affinity chromatograph (Tilbeurgh etc., FEBS Lett., 16:215 [1984])；Ion exchange chromatography (Goyal etc., Bio.Technol., 36:37 [1991]；Fliess etc., Eur.J.Appl.Microbiol.Biotechnol., 17:314 [1983]；Bhikhabhai etc., J.Appl.Biochem., 6:336 [1984]；With Ellouz etc., Chromatogr., 396:307 [1987]), including make the material of apparatus high resolution carry out ion-exchange chromatography (refering to such as Medve etc., J.Chromatogr.A 808:153 [1998])；Hydrophobic interaction chromatography (Tomaz and Queiroz, J.Chromatogr.A 865:123 [1999]；Two-phase partitioning (Brumbauer etc., Bioseparation 7:287 [1999])；Ethanol precipitates；Anti-phase HPLC；Chromatography on silicon dioxide or cation exchange resin (such as DEAE)；Chromatofocusing；SDS-PAGE；Ammonium sulfate precipitation；With Gel filtration is (such asG-75)。

In some preferred embodiments, detergent additives is used without purification from other components of culture medium Protein.In some such embodiments, concentrate described culture medium simply, then without further from nutrient media components This protein of middle purification and use.

Compared with containing the equivalent Bacillus host cell not changing (such as wild type) bglC gene, use place of the present invention The protein compositions that chief cell produces typically contains the cellulase of minimizing.

In one embodiment, the cellulase content of compositions by adding cellulosic polymerization by said composition Thing solution (such as containing the solution of 1% carboxymethyl cellulose (CMC)) is measured the viscosity-modifying of this solution afterwards and is assessed.Such Method (refers to such as Manning, Biochem.Biophys.Meth., 5:189-202 [1981] known to being；And Sheperd, Biochem.J., 193:67-74 [1981]).Can be used for the enforcement below of a kind of Exemplary viscosity determination test of the present invention Example provides.

In some embodiments comprising present protein compositions, there is with use the bud not changing bglC gene The minimizing that the equivalent proteins compositions that spore bacilli-cell produces causes is compared, and the present composition can result in less than 80% The cellulosic material solution viscosity of (being such as less than 50%, less than 30%, less than 20% or less than 10%) reduces.Real at some Executing in scheme, present protein compositions does not make the viscosity of cellulosic material solution produce detectable reduction, this In the case of this protein compositions be considered as the protein compositions of " cellulose-less enzyme ".

The protein compositions of cellulose-less enzyme can be used for multiple situation, includes but are not limited to laundry detergent, especially It it is the detergent containing cellulosic polymer.In some embodiments, the protein group of cellulose-less enzyme of the present invention is comprised The laundry detergent of compound comprises the surfactant of about 1% to 80% (such as 5% to 50%) (by weight).Real at some Executing in scheme, described surfactant is nonionic surfactant, and in other embodiments, it is cationic Surfactant, in other embodiments, it is anionic surfactant, and in other embodiments, it is Zwitterionic surface-active agent, in other embodiments, it comprise these surfactants any combination (such as cloudy from Son and the mixture of nonionic surfactant).Exemplary surfactants includes but are not limited to alkylbenzenesulfonate (ABS), including linear alkylbenzene sulfonate (LAS) and linear alkylsulfonic acids sodium, alkyl phenoxypolyethoxy ethanol (such as nonyl benzene Epoxide ethoxylate or nonyl phenol), diethanolamine, triethanolamine and monoethanolamine.Live in the surface that can be used for laundry detergent Other descriptions of property agent are provided in United States Patent (USP) No.3,664,961,3,919,678,4,222,905 and 4,239,659.

The laundry detergent of the enzyme comprising cellulose-less enzyme of the present invention can be in any form (such as solid, liquid, gel Deng) use.In some embodiments, described laundry detergent also comprises buffer agent, such as sodium carbonate, sodium bicarbonate or washing Detergent builders, bleach, bleach-activating, enzyme, enzyme stabilizers, suds booster, foam inhibitor, anti-tarnishing agent, corrosion inhibitor, dirt help Suspension, Soil Release Agents, antibacterial, pH adjusting agent, non-builder alkaline source, chelating agen, organic or inorganic filler, solvent, Hydrotropic agent, fluorescent whitening agent, dyestuff or spice.

As described above, in some embodiments, laundry detergent contains cellulosic polymer (as cellulose is polymerized Thing) or the cellulosic polymer of modification.Suitably cellulosic polymer include but are not limited to anion modified cellulose, The cellulose of the cellulose that nonionic is modified, cation modified cellulose, hybrid ion modification and mixture thereof, and fiber Element, cellulose ether, cellulose esters, cellulose amides, methylcellulose, carboxymethyl cellulose, ethyl cellulose, hydroxy ethyl fiber Element, hydroxypropyl methyl cellulose, ester carboxymethyl cellulose and any mixture thereof.In some embodiments, modified fiber Element ether polymer (refering to such as United States Patent (USP) No.6,833,347) or other cellulosic polymer (refer to such as United States Patent (USP) 5, 009,800 and 4,661,267) can be used for the present invention.In some embodiments, laundry detergent contains the most about The cellulosic polymer of 0.1% to 8% (e.g., from about 0.5% to 4% or about 1% to 3%).

Experiment

Following example are that those of ordinary skill in the art provide about the complete public affairs how implementing and using the present invention Open and describe, and be not intended to limit the scope of its invention that the present inventor is thought, and also do not mean that following experiment is institute The experiment completely or only having carried out.Do the best and ensured the accuracy of numeral (such as amount, temperature etc.) used, but some experiments Including error and deviation are also considered as.Except as otherwise noted, otherwise mark is mark by weight, and molecular weight is weight average mark Son amount, temperature is degree Celsius, and pressure is atmospheric pressure or close to atmospheric pressure.

In following experiment disclosure, employ following abbreviation: DEG C (degree Celsius)；Rpm (revolutions per minute)；H₂O (water)；Aa (aminoacid)；Bp (base pair)；Kb (kilobase to)；KD (kilodalton)；Gm (gram)；μ g and ug (microgram)；mg (milligram)；Ng (nanogram)；μ l and ul (microlitre)；Ml (milliliter)；Mm (millimeter)；Nm (nanometer)；μm and um (micron)；M (mole)； MM (mM)；μM and uM (micromole)；U (unit)；V (volt)；MW (molecular weight)；Sec (second)；Min (minute)；H and hr (hour)；OD₂₈₀(optical density of 280nm)；OD₄₀₅(optical density of 405nm)；OD₆₀₀(optical density of 600nm)；PAGE (poly-third Acrylamide gel electrophoresis)；LAS (dodecyl sodium sulfate)；SDS (sodium lauryl sulphate)；Tris (three (methylol) amino first Alkane).

Embodiment 1

Build bglC:: spectinomycin bacterial strain

The general strategy of disappearance bglC gene is shown in Figure 1A-B.In short, expand the upper of bglC gene flank by PCR Trip and downstream DNA fragment, and link together in the carrier.This Insert Fragment is opened by restriction endonuclease digestion, And connect by the spectinomycin box that flank is loxP site.Digested linear for gained plasmid by restriction endonuclease Change, and be transformed in bacillus cereus.By selecting with the antimicrobial labelling (spectinomycin) introduced, pass through double crossing over Event achieves the displacement of genes of interest.As bglC, with spectinomycin resistance gene permutation encoding district, use afterwards Identify the Cre recombinase in flank loxP site, this spectinomycin resistance gene ring is gone out.

Utilize primer described in strategy described in WO 03/083125 and table 1, use round pcr, constructed dna construct.

Restriction site is by following name: XbaI is TCTAGA (SEQ ID NO:20)；BamHI is GGATCC (SEQ ID NO:21)；SacI is GAGCTC (SEQ ID NO:22)；Asp718 is GGTACC (SEQ ID NO:23)；PstI is CTGCAG (SEQ ID NO:24), HindIII is AAGCTT (SEQ ID NO:25).

In the method, use 150 μ L Eppendorf pipes carry out 100 μ L PCR reaction, in described pipe containing 84 μ L water, 10 μ L PCR buffer, 1 every kind of primer of μ L (that is, BglC SacI UF and BglC BamH1 UR, or loxPBamH1F and LoxPBamH1R), 2 μ L dNTP, 1 μ L DNA profiling (such as wild type Bacillus chromosome or control plasmid) and 1 μ L DNA Polymerase.The archaeal dna polymerase used includes Taq Plus exo+ polymerase and Herculase (Stratagene).Reaction Following procedure is used to carry out in Hybaid PCRExpress thermal cycler.First by sample 94 DEG C of heating 5 minutes, then It is cooled to 50 DEG C of holdings.Archaeal dna polymerase is added at this point.25 amplification cycles respectively by 95 DEG C 1 minute, 50 DEG C 1 minute and 72 DEG C 1 Minute constitute.Final 72 DEG C guarantee fully extended in 10 minutes.Sample is maintained at 4 DEG C to analyzing.This reaction obtains flank DNA box (the most about 1kb) and 5 ' and 3 ' ends are with the loxP spectinomycin box of BamHI restriction site.

After PCR completes, by each for each reactant 10 μ L on Invitrogen 1.2% agarose E-gel with 60 volts electricity Swim 30 minutes, to check the existence of correct size strip.All gel electrophoresis described herein all use these conditions.If deposited At band, then use Qiagen according to the explanation of manufacturerIt is remaining instead that PCR purification kit carrys out purification Answer thing, then with suitable Restriction Enzyme to cutting.Digestion is carried out 1 hour at 37 DEG C, and 20 μ L reactants are by 9 μ L water, 2 μ L10 × BSA, 2 suitable for μ the L restricted buffer of NEB (according to 2000-01 NEB catalogue and technical data), 5 μ L templates and 1 μ L Every kind of Restriction Enzyme composition.Such as, with SacI and BamHI in NEB (New England BioLabs) restricted buffer B Cutting bglC fragment upstream.Qiagen is used by the gel purified fragment through digestion the explanation according to manufacturerGel extraction kit is extracted.

Explanation according to manufacturer uses Takara connect test kit or use T4 DNA ligase (reaction content: every Plant each 5 μ L of Insert Fragment, 1 pUC19 plasmid cleaved for μ L, 3 μ LT4DNA ligase buffer and 1 μ L T4 DNA ligase), In two steps fragment is connected in plasmid vector.First, cleaved upstream and segments downstream are overnight connected at 15 DEG C Tap into the pUC19 plasmid of the most SacI digestion gel-purified (refering to such as Yanisch-Reman etc., Gene33:103-119 [1985]), in the single restriction enzyme site in polylinker, connect in total BamHI site to re-form cyclic plasmid. The One Shot using manufacturer converts scheme and the Plastid transformation of this recirculation enters the competence " Top of Invitrogen 10 " in Bacillus coli cells.

With 1.5% agar (LA) add 50 μ g/ml Carbenicillins solidifications, containing X-gal (Sigma) for blue white sieve Choosing, in Luria-Bertani (LB) culture medium select transformant.Picked clones and at 37 DEG C at 5mL Luria Bertani Culture fluid (LB) adds overnight incubation in 50 μ g/ml Carbenicillins, uses Qiagen'sPreparation examination in a small amount Agent box separation quality grain.The restriction analysis using SacI to carry out confirms the existence of 2kb Insert Fragment.At the most above-mentioned digestion reaction (replacing the second Restriction Enzyme with 1 extra μ L water) cuts through with BamHI and confirms that the plasmid with this Insert Fragment is to incite somebody to action Its linearisation, with the phosphatase of the 1 little Intestinum Bovis seu Bubali of μ L or shrimp 37 DEG C process 1 hour to prevent from being cyclized again, and connect through BamHI digest The Spectinomycin resistance box that flank is loxP site.This connection mixture is transformed in escherichia coli Top 10 cell, And on the LA flat board containing 100 μ g/ml spectinomycins, select bacterium colony.As carried out restriction analysis above by with BamHI Confirm that the labelling in institute's separation quality grain inserts.Before being transformed in bacillus subtilis, with ScaI by plasmid linearization, with really Protect double cross-over event.

Embodiment 2

Viscosimetric analysis is tested

Cell is cultivated in be suitable to the 250ml band baffle plate conical flask of culture medium of production subtilisin containing 50mL (refering to Christianson etc., Anal.Biochem., 223:119-129 [1994]；And Hsia etc., Anal.Biochem., 242:221-227 [1996]).Inoculate this culture medium with 8 hours 5mL cultures of 50 μ L, and cultivate 40 hours simultaneously at 37 DEG C With 250 RPM shakes.Cultivate host A (-bglC), host B (-bglC), host C (-bglS) and Host D (-bglS) culture 48 hours.

Little from each shaking flask, 1ml sample is taken constantly 37.It is centrifuged off cell, 60 μ L of supernatant liquid are carried out viscosimetric analysis Test.Little t=0, t=22 hour and t=120 measure viscosity constantly.Use water as comparison.

Make to measure using the following method viscosity: for each experiment, (carboxymethyl is fine by 1% cellulosic material of 2ml volume Dimension element) disperse in profound hypothermia bottle.60 μ L sample are added in the cellulosic material of 2ml volume.After soft mixing, suitable When the scheduled time (such as hatching 20 hours) sampling be analyzed.For analyzing, 500 μ L sample and substrate are sucked viscosity In the specimen cup of meter (BrookfieldLV DVIII Cone&Plate viscometer, the CP40 water-bath with being set to 25 DEG C), and Programming viscometer so that SSN (arranging speed) is 16 RPM, WTI (waiting time) is set to 30 seconds (Rheocalc software). After 30 seconds, show summary sheet.

These results measured provide at Fig. 2.For contact the comparison of 1% cellulosic material, host A (-bglC) and For host's B (-bglC) sample, in experimentation, do not observe that viscosity declines, represent in sample and there is not cellulase activity Property.As represented by the decline of viscosity, the host strain of the bglS genome sequence with disappearance demonstrates significant cellulose Enzymatic activity.

Embodiment 3

Build bglC:: the spectinomycin bacterial strain producing protease

Test following bacterial strain in this experiment: produce subtilisin bacillus subtilis strain and with Insert the identical bacterial strain of the bglC of inactivation.

Bacterial strain to be tested constructed as below.It is used for the chromosomal DNA from the bacterial strain producing subtilisin and converts SC6 Bacterial strain (spoIIE, amyE, aprE Pxyl:comK).Containing chloromycetin (5 μ g/ml) and the L agar plate of 1.6% skimmed milk Upper selection transformant.The existence of subtilisin is confirmed by the haloing produced on the flat board containing skimmed milk.Gained The bacterial strain MDT04-250 chromosomal DNA from the bacterial strain with the bglC gene inserting inactivation converts, and containing strong Transformant is selected on the L agar plate of miromycin (100 μ g/ml).By to chloromycetin (5 μ g/ml) and spectinomycin (100 μ g/ Ml) resistance confirms new strains MDT05-28.For obtaining protease, expanding both bacterial strains, this is by containing it successively There is chloromycetin (10 μ g/ml) then to contain on the L agar plate of chloromycetin (25 μ g/ml) to cultivate and carry out.At 250mL band baffle plate Conical flask in, in the shaking flask containing 25ml LB (Difco), glucose (0.1%) and chloromycetin (25 μm/ml), cultivate this A bit through the bacterial strain of amplification.Shaking flask is hatched at 37 DEG C and shakes with 280rpm, at OD simultaneously₅₅₀When being 0.8, by 1mL culture with 0.5ml 30% glycerol mixes and is frozen in-70 DEG C.To solve the 40ml FNII in 30 μ L inoculation 250ml triangular flasks in frozen pipe Culture medium.Each bacterial strain uses two bottles.Shaking flask is hatched a couple of days with 37 DEG C under 280rpm shakes, collects supernatant samples and carry out Viscosity test and subtilisin analysis.

After different time (such as 16,48 and 68 hours), from liquid culture, supernatant is gathered in the crops in incubation, and At 25 DEG C containing 0.3 mM N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-paranitroanilinum (Vega Biochemicals), 0.1M Tris, in the solution of pH 8.6 as aforementioned mensuration subtilisin (refering to Estell etc., J.Biol.Chem., 260:6518-6521 [1985]).This algoscopy is measured owing to hydrolysis and the release of paranitroanilinum cause The raising of 410nm absorbance per minute.These bacterial strains produce the subtilisin of equal quantities at an equal rate.

16,48 or 68 are little constantly from parent strain MDT04-250 or bglC::spec bacterial strain (MDT05-28) after inoculation Shaking flask in take supernatant samples, and to hatch 1,24 or 48 little with cellulosic substrate (4.5ml 5%CMC+0.5ml supernatant) Time.Being declined by the viscosity of cellulosic substrate and weigh the cellulase activity to cellulosic substrate, above-mentioned viscosity declines basis The guidance of manufacturer uses software by rheometry (as described above).Data are to compare (dH₂O) percentage ratio of reading comes Represent.The sample deriving from MDT05-28 shaking flask does not shows that the viscosity of cellulosic substrate reduces, and from all samples of MDT04-250 Product reduce the viscosity of cellulosic substrate the most rapidly.This shows that the inactivation of bglC prevents the activity to cellulosic substrate.These The result of determination test is shown in Fig. 3.These results show, have the expression subtilisin of the bglC gene of destroyed The culture supernatants of host's Bacillus strain does not shows significant cellulase activity.

Although illustrate and described specific embodiments of the present invention, but to those skilled in the art Change and amendment, without departing from the spirit and scope of the present invention it will be apparent that multiple other can be carried out.Therefore, appended Claims are intended to all these change within the scope of the present invention and amendment.

The all patents mentioned in this specification and publication represent the level of those skilled in the art of the invention.Institute Having patent and publication to be expressly incorporated herein the most in way of reference, its degree is equal to every single publication and the most specifically and individually refers to Bright it is incorporated in way of reference.

Notwithstanding the preferred embodiments of the invention, but for those of ordinary skill in the art it will be apparent that Disclosed embodiment can be carried out multiple amendment, and these amendments are the most within the scope of the present invention.

Those skilled in the art are fully aware of, and the present invention is adapted to realize described by the purpose of the present invention acquisition very much Result and advantage and the result of its inherence and advantage.Compositions as herein described and method represent preferred embodiment, are to show Example, and be not intended to limit the scope of the present invention.To those skilled in the art it will be apparent that can be to this paper institute State invention and carry out a variety of substitutions and modifications, without departing from scope and spirit of the present invention.

Aptly, the invention of description exemplified here can lack any key element not specifically disclosed herein, limit bar Implement in the case of part.The term used and statement are with being described term and unrestricted, and the use of these terms and statement is also It is not intended to get rid of any equivalent of illustrated and described feature or its part, but it is recognized that, protect at application claims Multiple amendment can be had within the scope of protecting.Although it should therefore be understood that the present invention is by preferred embodiment and optional feature Carry out concrete disclosure, but concepts disclosed herein can have been modified and change by those skilled in the art, these amendments It is all considered within the scope of the invention defined in appended claims with change.

Herein the present invention has been carried out wide in range and general description.Fall into every kind of bottom within disclosed upperseat concept Concept and subgroup all constitute the part of the present invention.This include sub conditione or negative sense limiting factor, the upper of the present invention is retouched Stating, to remove any theme from this upperseat concept, no matter this theme being excluded the most specifically is mentioned.

Sequence table

<110>Danisco Us Inc. Genencor Divisi (Danisco US, Inc., Genencor Division)

<120>Cellulase-Free Enzyme Compositions and Host Cells for Producing

the Same

<130>GC930-PCT

<140>PCT/US2007/020851

<141>2007-09-26

<150>US 60/849,982

<151>2006-10-06

<160>26

<170>PatentIn version 3.4

<210>1

<211>1478

<212>DNA

<213>Bacillus spec (Bacillus sp.)

<400>1

atgaaacggt caatctctat ttttattacg tgtttattga ttacgttatt gacaatgggc 60

ggcatgatag cttcgccggc atcagcagca gggacaaaaa cgccagtagc caagaatggc 120

cagcttaata aaaggtacac agctcgttaa ccgagacggt aaagcggtac agctgaaggg 180

gatcagttca cacggattgc aatggtatgg agaatatgtc aataaagaca gcttaaaatg 240

gctgagagat gatgggtatc accgttttcc gtgcagcgat gtatacggca gatggcggtt 300

atattgacaa cccgtccgtg aaaaataaag taaaagaagc ggttgaagcg gcaaaagagc 360

ttgggatata tgtcatcatg atggcatatc ttaaatgacg gtaatccaaa ccaaaataaa 420

gagaaggcaa aagaattctt caaggaaatg tcaagccttt acggaaacac gccaaacgtc 480

atttatgaaa ttgcaaacga accaacggga tgtgaactgg aagcgtgata ttaaaccata 540

tgcggaagaa gtgatttcag ttatccgcaa aaatgatcca gacaacatca tcattgtcgg 600

aaccggtaca tggagccagg atgtgaatga tctgcgatga ccagctaaaa gatgcaaacg 660

ttatgtacgc acttcatttt tatgccggca cgcacggcca atttttacgg gataaagcaa 720

actatgcact cagcaaagga gcacctattt ttgtgacgag tgggaacaag cgacgcgtct 780

ggcaatggcg gtgtattcct tgatcaatcg agggaatggc tgaaatatct cgacagcaag 840

accattagct gggtgaactg gaatctttct gataagcagg aatatcctcg ctttaaagcc 900

gggggcatct aaaacaggcg gctggcggtt gtcagattta tctgcttcag gaacattcgt 960

tagagaaaac attctcggca ccaaagattc gacgaaggac attcctgaac gccatcaaag 1020

ataaacccac acaggaaaat ggtatttctg tacagtacag agcaggggat gggagtatga 1080

acagcaacca aatccgtccg cagcttcaaa taaaaaataa cggcaatacc acggtgattt 1140

aaagatgtca ctgcccgtta ctggtataaa gcgaaaaaca aaggccaaaa ctttgactgt 1200

gactacgcgc agattggatg cggcaatgtg acacacaagt ttgtgacgtt gcataaacca 1260

agcaaggtgc gatacctatc tggaacttgg atttaaaaac ggaacgttgg caccgggagc 1320

aagcacaggg aatattcagc tccgtcttca caatgatgac tggagcaatt atgcacaaag 1380

cggcgatatt cctttttaaa tcaaatacgt ttaaaacaac gaaaaaaatc acattatatg 1440

atcaaggaaa actgatttgg ggaacagaac caaattag 1478

<210>2

<211>493

<212>PRT

<213>Bacillus spec

<400>2

Met Lys Arg Ser Ile Ser Ile Phe Ile Thr Cys Leu Leu Ile Thr Leu

1 5 10 15

Leu Thr Met Gly Gly Met Ile Ala Ser Pro Ala Ser Ala Ala Gly Thr

20 25 30

Lys Thr Pro Val Ala Lys Asn Gly Gln Leu Ser Ile Lys Gly Thr Gln

35 40 45

Leu Val Asn Arg Asp Gly Lys Ala Val Gln Leu Lys Gly Ile Ser Ser

50 55 60

His Gly Leu Gln Trp Tyr Gly Glu Tyr Val Asn Lys Asp Ser Leu Lys

65 70 75 80

Trp Leu Arg Asp Asp Trp Gly Ile Thr Val Phe Arg Ala Ala Met Tyr

85 90 95

Thr Ala Asp Gly Gly Tyr Ile Asp Asn Pro Ser Val Lys Asn Lys Val

100 105 110

Lys Glu Ala Val Glu Ala Ala Lys Glu Leu Gly Ile Tyr Val Ile Trp

115 120 125

His Ile Leu Asn Asp Gly Asn Pro Asn Gln Asn Lys Glu Lys Ala Lys

130 135 140

Glu Phe Phe Lys Glu Met Ser Ser Leu Tyr Gly Asn Thr Pro Asn Val

145 150 155 160

Ile Tyr Glu Ile Ala Asn Glu Pro Asn Gly Asp Val Asn Trp Lys Arg

165 170 175

Asp Ile Lys Pro Tyr Ala Glu Glu Val Ile Ser Val Ile Arg Lys Asn

180 185 190

Asp Pro Asp Asn Ile Ile Ile Val Gly Thr Gly Thr Trp Ser Gln Asp

195 200 205

Val Asn Asp Ala Ala Asp Asp Gln Leu Lys Asp Ala Asn Val Met Tyr

210 215 220

Ala Leu His Phe Tyr Ala Gly Thr His Gly Gln Phe Leu Arg Asp Lys

225 230 235 240

Ala Asn Tyr Ala Leu Ser Lys Gly Ala Pro Ile Phe Val Glu Gly Thr

245 250 255

Ser Asp Ala Ser Gly Asn Gly Gly Val Phe Leu Asp Gln Ser Arg Glu

260 265 270

Trp Leu Lys Tyr Leu Asp Ser Lys Thr Ile Ser Trp Val Asn Trp Asn

275 280 285

Leu Ser Asp Lys Gln Glu Ser Ser Ser Ala Leu Lys Pro Gly Ala Ser

290 295 300

Lys Thr Gly Gly Trp Arg Leu Ser Asp Leu Ser Ala Ser Gly Thr Phe

305 310 315 320

Val Arg Glu Asn Ile Leu Gly Thr Lys Asp Ser Thr Lys Asp Ile Pro

325 330 335

Glu Thr Pro Ser Lys Asp Lys Pro Thr Gln Glu Asn Gly Ile Ser Val

340 345 350

Gln Tyr Arg Ala Gly Asp Gly Ser Met Asn Ser Asn Gln Ile Arg Pro

355 360 365

Gln Leu Gln Ile Lys Asn Asn Gly Asn Thr Thr Asp Leu Asp Val Thr

370 375 380

Ala Arg Tyr Trp Tyr Lys Ala Lys Asn Lys Gly Gln Asn Phe Asp Cys

385 390 395 400

Asp Tyr Ala Gln Ile Gly Cys Gly Asn Val Thr His Lys Phe Val Thr

405 410 415

Leu His Lys Pro Lys Gln Gly Ala Asp Thr Tyr Leu Glu Leu Gly Phe

420 425 430

Lys Asn Gly Thr Leu Ala Pro Gly Ala Ser Thr Gly Asn Ile Gln Leu

435 440 445

Arg Leu His Asn Asp Asp Trp Ser Asn Tyr Ala Gln Ser Gly Asp Tyr

450 455 460

Ser Phe Phe Lys Ser Asn Thr Phe Lys Thr Thr Lys Lys Ile Thr Leu

465 470 475 480

Tyr Asp Gln Gly Lys Leu Ile Trp Gly Thr Glu Pro Asn

485 490

<210>3

<211>37

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>3

acaaatgagc tcgctggagc attggatggc gcattcc 37

<210>4

<211>37

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>4

tgatctggat ccccttcgca tcattttggc tctacac 37

<210>5

<211>37

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>5

aaaactggat ccgggaacag aaccaaatta gttaagc 37

<210>6

<211>37

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>6

atggtagtcg acgcaaacgc ggctacaata tggctca 37

<210>7

<211>25

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>7

ttccgcggag ggccggccta ctata 25

<210>8

<211>25

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>8

catattcaca atgcgatggt agagg 25

<210>9

<211>25

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>9

ttatgcacaa agcggcgattattcc 25

<210>10

<211>22

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>10

atctcttgcc agtcacgtta cg 22

<210>11

<211>37

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>11

ggagtgtcta gaactgacca gcttccgtct ttccctg 37

<210>12

<211>37

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>12

actaacggat cccctgtaac tatcatcatc ttccctc 37

<210>13

<211>37

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>13

caaaaaggat ccgccaaatg tgaaagagcc tgctgca 37

<210>14

<211>37

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>14

agaggtgagc tcaccgctga ttcccgctat gatcgcc 37

<210>15

<211>27

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>15

caatatacac aatacagtgc tgaaagc 27

<210>16

<211>25

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>16

gcgggaatag cgatgcttgg ttcgg 25

<210>17

<211>25

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>17

gatgaacttg tggaatggca cgggt 25

<210>18

<211>30

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>18

tcgacggtat cgataagctg gatccataac 30

<210>19

<211>32

<212>DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400>19

gcctaggatg catatgggat ccgcataact tc 32

<210>20

<211>6

<212>DNA

<213>artificial sequence

<220>

<223>restriction site synthesized

<400>20

tctaga 6

<210>21

<211>6

<212>DNA

<213>artificial sequence

<220>

<223>restriction site synthesized

<400>21

ggatcc 6

<210>22

<211>6

<212>DNA

<213>artificial sequence

<220>

<223>restriction site synthesized

<400>22

gagctc 6

<210>23

<211>6

<212>DNA

<213>artificial sequence

<220>

<223>restriction site synthesized

<400>23

ggtacc 6

<210>24

<211>6

<212>DNA

<213>artificial sequence

<220>

<223>restriction site synthesized

<400>24

ctgcag 6

<210>25

<211>6

<212>DNA

<213>artificial sequence

<220>

<223>restriction site synthesized

<400>25

aagctt 6

<210>26

<211>4

<212>PRT

<213>artificial sequence

<220>

<223>reagent synthesized

<220>

<221>VARIANT

<222>(1)..(2)

<223>L-Ala

<220>

<221>VARIANT

<222>(3)..(3)

<223>L-Pro

<220>

<221>VARIANT

<222>(4)..(4)

<223>L-Phe

<400>26

Ala Ala Pro Phe

1

Claims (20)

1. the Bacillus host cell of the cellulose-less enzyme of restructuring, it comprises the base containing the bglC gene inactivated through restructuring Because of group, wherein said cell also comprises the recombinant nucleic acid for producing secreting type detergent additives protein.

2. the recombinated bacillus host cell of claim 1, the bglC gene of wherein said inactivation comprises disappearance, inserts, replaces Change or reset.

3. the recombinated bacillus host cell of claim 1, wherein said bacillus cell is Bacillus licheniformis (B.licheniformis), bacillus subtilis, Bacillus clausii (B.clausii), Alkaliphilic bacillus (B.alkalophilus) or salt tolerant bacillus cereus (B.halodurans) cell.

4. the recombinated bacillus host cell of claim 1, wherein said secreting type detergent additives protein is for being selected from The enzyme of protease, amylase, pectate lyase, acyltransferase, arylesterase and lipase.

5. the recombinated bacillus host cell of claim 4, wherein said enzyme is protease.

6. the recombinated bacillus host cell of claim 5, wherein said protease is subtilisin.

7. the recombinated bacillus host cell of claim 1, wherein said bglC gene code and SEQ ID NO:2 have to The polypeptide of few 80% homogeneity.

8. cell culture, it comprises:

Culture medium；With

The recombinated bacillus host cell of claim 1.

9. it is used for producing a secreting type detergent additives method of protein, comprising:

The cell culture of claim 8 is maintained under conditions of being suitable to produce described secreting type detergent additives protein.

10. the method for claim 9, also includes:

Described secreting type detergent additives protein is reclaimed from described culture medium.

The method of 11. claim 10, also includes combining described secreting type detergent additives protein with laundry detergent.

The method of 12. claim 9, wherein said secreting type detergent additives protein is subtilisin.

13. cell cultures produced by the method for claim 9 or its culture medium are at the protein preparing cellulose-less enzyme Purposes in compositions, wherein said cell culture or culture medium comprise described secreting type detergent additives protein.

The purposes of 14. claim 13, wherein said compositions comprises the secreting type detergent additives produced by bacillus cereus Protein, and it is characterized in that said composition does not have detectable cellulase activity.

15. cell culture or its culture medium produced by the method for claim 9 are washed in the laundry preparing cellulose-less enzyme Washing the purposes in agent, wherein said cell culture or culture medium comprise described secreting type detergent additives protein.

The purposes of 16. claim 15, wherein said detergent contains cellulosic polymer.

17. methods building cell, it includes

A) the first recombinant nucleic acid is introduced in bacillus cell, so that described recombinant nucleic acid and the bglC gene weight of described cell Group, causes bglC gene inactivation；With

B) being introduced in described cell by the second recombinant nucleic acid, it provides secreting type detergent additives protein expression,

Wherein, described cell produces the detergent additives protein of cellulose-less enzyme.

The method of 18. claim 17, wherein said first recombinant nucleic acid is inserted in described bglC gene.

The method of 19. claim 17, wherein said first recombinant nucleic acid causes at least one part of described bglC gene to lack Lose.

The method of 20. claim 17, wherein said secreting type detergent additives protein is subtilisin.